Toll-like receptor agonist formulations and their use

ABSTRACT

The present invention is directed generally to stable formulations of a TLR agonist preferably a TLR7 or a TLR8 agonist, for use in the treatment of cancer, preferably solid tumors and lymphomas. Specifically, the present invention is directed to stable formulations of up to 50 mg/ml of a TLR agonist which comprise a cyclodextrin.

1. RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/533,596 (allowed), filed Jul. 31, 2009, which claims priorityunder 35 U.S.C. 119(e) to U.S. Application No. 61/137,694, filed Aug. 1,2008. The entire contents of the above-identified applications arehereby incorporated by reference.

2. FIELD OF THE INVENTION

The present invention is directed to pharmaceutical formulations oftoll-like receptor (TLR) agonists and their use.

3. BACKGROUND OF THE INVENTION

Toll-like receptors (TLRs) are a family of type I transmembrane proteinswhose in vivo activation initiates an innate immune response involvingspecific cytokines, chemokines and growth factors. While all TLRs canactivate certain intracellular signaling molecules such as nuclearfactor kappa beta (NF-kB) and mitogen activated protein kinases (MAPkinases), the specific set of cytokines and chemokines released appearsto be unique for each TLR. TLR7, 8, and 9 comprise a subfamily of TLRswhich are located in endosomal or lysosomal compartments of immune cellssuch as dendritic cells and monocytes. Specifically, TLR7 is expressedby plasmacytoid dendritic cells and to a lesser extent by monocytes, andTLR8 is expressed by monocytes as well as by monocyte-derived dendriticcells and myeloid dendritic cells. This subfamily mediates recognitionof microbial nucleic acids, such as single stranded RNA. Agonists ofTLR7 and/or TLR8 stimulate the production of various inflammatorycytokines including interleukin-6, interleukin-12, tumor necrosisfactor-alpha, and interferon-gamma. Such agonists also promote theincreased expression of co-stimulatory molecules such as CD40, CD80, andCD86, major histocompatibility complex molecules, and chemokinereceptors. The type I interferons, IFNα and IFNβ, are also produced bycells upon activation with TLR7/8 agonists.

Small, low-molecular weight (less than 400 Daltons) syntheticimidazoquinoline compounds which resemble the purine nucleotidesadenosine and guanosine were the first TLR7 and TLR8 agonists to beidentified. A number of these compounds have demonstrated anti-viral andanti-cancer properties. For example, the TLR7 agonist imiquimod(ALDARA™) was approved by the U.S. Food and Drug Administration as atopical agent for the treatment of skin lesions caused by certainstrains of the human papillomavirus. Imiquimod may also be useful forthe treatment of primary skin cancers and cutaneous tumors such as basalcell carcinomas, keratoacanthomas, actinic keratoses, and Bowen'sdisease. The TLR7/8 agonist resiquimod (R-848) is being evaluated as atopical agent for the treatment of human genital herpes.

4. SUMMARY OF THE INVENTION

The present invention is directed generally to formulations ofpharmaceutical compositions containing a benzo[b]azepine TLR agonist foruse in the treatment of cancer, preferably solid tumors and lymphomas,and for other uses including the treatment of certain skin conditions ordiseases, such as atopic dermatitis, the treatment of infectiousdiseases, preferably viral diseases, and for use as adjuvants invaccines formulated for use in cancer therapy and in the treatment ofinfectious diseases. Specifically, the present invention is directed tostable formulations of a benzo[b]azepine TLR agonist, preferably a TLR7or a TLR8 agonist. In preferred embodiments, the benzo[b]azepine TLR7 orTLR8 agonist is used for the treatment of cancer and the cancer isselected from the group consisting of ovarian cancer, breast cancer,head and neck cancer, renal cancer, bladder cancer, hepatocellularcancer, and lymphoma.

Preferably, the benzo[b]azepine TLR agonist is formulated at aconcentration of from about 0.5 mg/ml to about 50 mg/ml, from about 1mg/ml to about 40 mg/ml, or from about 2 mg/ml to about 15 mg/ml. Incertain embodiments, the benzo[b]azepine TLR agonist is formulated at aconcentration of from about 0.5 mg/ml to about 10 mg/ml, from about 0.5mg/ml to about 8 mg/ml, from about 0.5 mg/ml to about 6 mg/ml, fromabout 0.5 mg/ml to about 4 mg/ml, or from about 0.5 mg/ml to about 2mg/ml. In certain embodiments, the benzo[b]azepine TLR agonist isformulated at a concentration of about 0.5 mg/ml, about 1 mg/ml, about 2mg/ml, about 4 mg/ml, about 6 mg/ml, about 8 mg/ml, about 10 mg/ml,about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 40mg/ml, or about 50 mg/ml. Preferably, the formulation comprises about1-30%, 5-15%, or 5-10% weight/volume (w/v) of a cyclodextrin, preferablya β-cyclodextrin, and most preferably sulfobutylether β-cyclodextrin. Incertain embodiments, the formulation comprises 1%, 5%, 10%, 15%, 20%,25%, or 30% w/v of a cyclodextrin, preferably a β-cyclodextrin, and mostpreferably sulfobutylether β-cyclodextrin. In a particular embodiment,the formulation is an aqueous solution comprising the benzo[b]azepineTLR agonist at a concentration of at least 2 mg/ml. In a furtherembodiment, the formulation comprises 15% w/v of a cyclodextrin,preferably a β-cyclodextrin, and most preferably sulfobutyl etherβ-cyclodextrin. In preferred embodiments, the formulation is suitablefor injection in a mammal, preferably a human. In particularembodiments, injection is by a subcutaneous route, an intramuscularroute, or transdermal route. In certain embodiments, the formulation issuitable for intravenous administration.

In certain embodiments, the pH of the benzo[b]azepine TLR agonistformulation is acidic (meaning less than 7). Preferably, the pH of thebenzo[b]azepine TLR agonist formulation is from about 5.0 to about 7,preferably from about 5.5 to about 6.5. In a particular preferredembodiment, the pH is 6.5. In one embodiment, the benzo[b]azepine TLRagonist formulation is a solid, a liquid or lyophilized formulationsuitable for injection in a mammal, preferably a human. In oneembodiment, the formulation is sterile. In certain embodiments, thepresent formulations are stable at temperatures ranging from about 20 to25° C. for at least 1 week and the formulation is stable for at least 2weeks at about 2 to 8° C.

The present invention also provides lyophilized formulations of abenzo[b]azepine TLR agonist which when reconstituted in aqueous solutionare substantially soluble. Preferably, the reconstituted formulation issuitable for injection in a mammal, preferably a human. In particularembodiments, injection is by a subcutaneous route, an intramuscularroute, or transdermal route. In certain embodiments, the formulation issuitable for intravenous administration.

In certain preferred embodiments, the benzo[b]azepine TLR agonistformulation comprises one or more of the following benzo[b]azepine TLRagonists: (1E, 4E)-ethyl2-amino-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxylate; (1E,4E)-2-amino-N,N-bis(2-methoxyethyl)-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-N,N-diethyl-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-8-(perfluoroethyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-N-ethyl-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-8-(perfluoroethyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide;(1E, 4E)-ethyl2-amino-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxylate;(1E, 4E)-ethyl2-amino-8-(4-(methoxycarbonyl)phenyl)-3H-benzo[b]azepine-4-carboxylate;(1E, 4E)-ethyl2-amino-8-(4-(methylcarbamoyl)phenyl)-3H-benzo[b]azepine-4-carboxylate;(1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamide;and pharmaceutically acceptable salts thereof. In a particularembodiment, the benzo[b]azepine TLR agonist is (1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamideand pharmaceutically acceptable salts thereof.

The present invention further provides methods for the treatment ofcancer by administering to a subject, preferably a human subject, abenzo[b]azepine TLR agonist formulation of the present invention, whichcontains a cyclodextrin. In a preferred embodiment, the benzo[b]azepineTLR agonist formulation is administered in combination with one or moreadditional treatment modalities, where the modalities are selected froma chemotherapeutic agent, a cytokine, an antibody, hormonal therapy, orradiation therapy. In one embodiment, the benzo[b]azepine TLR agonistformulation is administered as part of a regimen for the treatment of asolid tumor. In a further embodiment, the solid tumor is a form ofcancer selected from among ovarian cancer, breast cancer, head and neckcancer, renal cancer, bladder cancer, hepatocellular cancer, orlymphoma. In one embodiment, the benzo[b]azepine TLR agonist formulationis administered as part of a regimen for the treatment of a lymphoma. Inone embodiment, the lymphoma is Hodgkin's lymphoma. In anotherembodiment, the lymphoma is non-Hodgkin's lymphoma. In anotherembodiment, the benzo[b]azepine TLR agonist formulation is used as avaccine adjuvant for the treatment of cancer. In certain embodiments ofthe methods for the treatment of cancer, the benzo[b]azepine TLR agonistformulation is administered by injection or intravenously. In particularembodiments, injection is by a subcutaneous route, an intramuscularroute, or a transdermal route. In a particular embodiment, theformulation is administered by subcutaneous injection.

The present invention also provides methods for the treatment of atopicdermatitis by administering to a subject, preferably a human subject, abenzo[b]azepine TLR agonist formulation of the present invention, whichcontains a cyclodextrin. According to this embodiment, the route ofadministration is preferably subcutaneous or topical.

The present invention also provides methods for the treatment of aninfectious disease by administering to a subject, preferably a humansubject, a benzo[b]azepine TLR agonist formulation of the presentinvention, which contains a cyclodextrin. Preferably, the infectiousdisease is caused by a virus. In a particular embodiment, the virus ishepatitis C virus (HCV or HepC).

In certain embodiments of the methods for treating cancer or infectiousdisease, the benzo[b]azepine TLR agonist is administered to the subjectat a dose of about 0.02 to 10 mg/kg or about 0.04 to 5 mg/kg body weightof the subject. In certain embodiments, the benzo[b]azepine TLR agonistis administered at a dose of about 0.02 mg/kg, about 0.05 mg/kg, about 1mg/kg, about 2 mg/kg, or about 5 mg/kg. In certain further embodiments,the benzo[b]azepine TLR agonist formulation is administered to thesubject on a weekly or biweekly basis.

The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with a liquid or lyophilizedbenzo[b]azepine TLR agonist formulation of the invention for thetreatment of cancer or one or more symptoms thereof. Preferably, theformulation comprises about 1-30%, 5-15%, or 5-10% w/v of acyclodextrin, preferably a β-cyclodextrin, and most preferablysulfobutylether β-cyclodextrin. In certain embodiments, the formulationcomprises 2%, 5%, 10%, 15%, 20%, 25%, or 30% w/v of a cyclodextrin,preferably a β-cyclodextrin, and most preferably sulfobutyletherβ-cyclodextrin. In a particular embodiment, the formulation is anaqueous formulation of the benzo[b]azepine TLR agonist (1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamidecontaining a β-cyclodextrin, preferably sulfobutylether β-cyclodextrin.In another embodiment, the formulation is a lyophilized formulation of(1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamidecontaining a β-cyclodextrin, preferably sulfobutylether β-cyclodextrin.Preferably, the benzo[b]azepine TLR agonist is formulated at aconcentration of at least 2 mg/ml and the formulation, whether aqueousor a reconstituted lyophilized formulation, is suitable for subcutaneousinjection in a mammal, preferably a human.

The present invention also provides for a use of a formulation of abenzo[b]azepine TLR agonist, preferably a TLR7 or a TLR8 agonist in themanufacture of a medicament for treating cancer, an infectious disease,or atopic dermatitis in a subject. The cancer is selected from the groupconsisting of ovarian cancer, breast cancer, head and neck cancer, renalcancer, bladder cancer, hepatocellular cancer, and lymphoma.

In one embodiment, the TLR agonist is formulated at a concentration ofat least 2 mg/ml. Moreover, the formulation is suitable foradministration to the subject, where the subject is preferably a human,by injection and is by subcutaneous, intramuscular, or transdermalinjection. In certain embodiments, the TLR agonist is administered tothe subject at a dose of about 0.02 to 10 mg/kg, at a dose of about 0.04to 5 mg/kg. In certain further embodiments, the benzo[b]azepine TLRagonist formulation is administered to the subject on a weekly orbiweekly basis.

In a preferred embodiment, the benzo[b]azepine TLR agonist formulationis administered in combination with one or more additional treatmentmodalities, where the modalities are selected from a chemotherapeuticagent, a cytokine, an antibody, hormonal therapy, or radiation therapy.The present invention also provides methods for the treatment ofinfectious disease is caused by a virus, where the virus is a hepatitisvirus.

The above description sets forth rather broadly the more importantfeatures of the present invention in order that the detailed descriptionthereof that follows may be understood, and in order that the presentcontributions to the art may be better appreciated. Other objects andfeatures of the present invention will become apparent from thefollowing detailed description considered in conjunction with theexamples.

5. DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control.

The present invention provides stable formulations of benzo[b]azepineTLR agonists and methods of using the same. The benzo[b]azepine TLRagonists of the invention are preferably TLR7 or TLR8 agonists, or haveagonist activity for both TLR7 and TLR8. Benzo[b]azepine TLR agonistswhich can be formulated according to the present invention are describedin PCT International Application No. PCT/US2006/032098, filed Aug. 17,2006. In a preferred embodiment, the benzo[b]azepine TLR agonist is (1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamideand pharmaceutically acceptable salts thereof. The formulations of thepresent invention are suitable for use in methods for the treatment ofhuman diseases such as cancer and infectious diseases as describedherein.

TLR Agonists of the Invention

Benzo[b]azepine TLR agonists which can be formulated according to thepresent invention are described in PCT International Application No.PCT/US2006/032098, filed Aug. 17, 2006, published as WO 2007/024612 onMar. 1, 2007, the contents of which are incorporated herein in theirentirety. In a preferred embodiment, the benzo[b]azepine TLR agonist is(1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamideand pharmaceutically acceptable salts thereof.

Preferably, the benzo[b]azepine TLR agonists of the present inventionare compounds of Formula I:

and metabolites, solvates, tautomers, and pharmaceutically acceptablesalts and

prodrugs thereof, wherein:

Y is CF₂CF₃, CF₂CF₂R⁶, or an, aryl or heteroaryl ring, wherein said aryland heteroaryl rings are substituted with one or more groupsindependently selected from alkenyl, alkynyl, Br, CN, OH, NR⁶R⁷,C(═O)R⁸, NR⁶SO₂R⁷, (C₁-C₆ alkyl)amino, R⁶OC(═O)CH═CH₂—, SR⁶ and SO₂R⁶,and wherein said aryl and heteroaryl rings are optionally furthersubstituted with one or more groups independently selected from F, Cl,CF₃, CF₃O—, HCF₂O—, alkyl, heteroalkyl and ArO—;

R¹, R³ and R⁴ are independently selected from H, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶, and SO₂R⁶,

or R³ and R⁴ together with the atom to which they are attached form asaturated or partially unsaturated carbocyclic ring, wherein saidcarbocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶, and SO₂R⁶;

R² and R⁸ are independently selected from H, OR⁶, NR⁶R⁷, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶, and SO₂R⁶; R^(5a), R^(5b), and R^(5C)are independently H, F, Cl, Br, I, OMe, CH₃, CH₂F, CHF₂ or CF3;

and

R⁶ and R⁷ are independently selected from H, alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl andheteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶ _(R) ⁷,C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶, and SO₂R⁶,

or R⁶ and R⁷ together with the atom to which they are attached form asaturated or partially unsaturated heterocyclic ring, wherein saidheterocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶, and SO₂R⁶.

In certain embodiments, R¹, R³, and R⁴ are each hydrogen.

In certain embodiments, R^(5a), R^(5b), and R^(5C) are each hydrogen.

In certain embodiments of the compound of Formula I, R² is OR⁶. Incertain embodiments, R⁶ is alkyl, such as (1-4C)alkyl. In particularembodiments, R⁶ is ethyl.

In certain embodiments of the compound of Formula I, R² is NR⁶R⁷. Incertain embodiments, R⁶ and R⁷ are independently H, alkyl, such as(1-6C)alkyl, or heteroalkyl, such as (1-4C)alkoxy(2-4C)alkyl. Inparticular embodiments, R⁶ and R⁷ are independently H, ethyl, propyl, orCH₂CH₂OCH₃.

In certain embodiments of the compound of Formula I, Y is aryl, such asphenyl. In certain embodiments, said aryl is substituted with C(═O)R⁸,such as in para-R⁸C(═O)phenyl. In certain embodiments, R⁸ is OR⁶, NR⁶R⁷or heterocycloalkyl. In certain embodiments, R⁶ and R⁷ are independentlyH or alkyl, such as (1-6C)alkyl. In certain other embodiments, R⁶ and R⁷together with the nitrogen atom to which they are attached form a 4-6membered azacycloalkyl ring, such as pyrrolidinyl. In particularembodiments, Y is

In certain embodiments of said compound of Formula I, Y is CF₂CF₃.

In specific embodiments, the benzo[b]azepine TLR agonist is selectedfrom among the following compounds:

-   -   (1E, 4E)-ethyl        2-amino-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxylate;    -   (1E,        4E)-2-amino-N,N-bis(2-methoxyethyl)-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;    -   (1E,        4E)-2-amino-N,N-diethyl-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;    -   (1E,        4E)-2-amino-8-(perfluoroethyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide;    -   (1E,        4E)-2-amino-N-ethyl-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;    -   (1E,        4E)-2-amino-8-(perfluoroethyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide;    -   (1E, 4E)-ethyl        2-amino-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxylate;    -   (1E, 4E)-ethyl        2-amino-8-(4-(methoxycarbonyl)phenyl)-3H-benzo[b]azepine-4-carboxylate;    -   (1E, 4E)-ethyl        2-amino-8-(4-(methylcarbamoyl)phenyl)-3H-benzo[b]azepine-4-carboxylate;    -   (1E,        4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamide;        and pharmaceutically acceptable salts thereof.

5.1 Formulations

The present invention provides stable formulations of thebenzo[b]azepine TLR agonists described in Section 5.1. The formulationsof the invention are preferably suitable for pharmaceutical uses asdescribed in Sections 5.3. Most preferably, the formulations aresuitable for subcutaneous administration to a subject, preferably ahuman subject, but can be for administration by other means as describedin Section 5.4.

The benzo[b]azepine TLR agonist formulations of the present inventioncomprise one or more pharmaceutically acceptable excipients. The termexcipient as used herein broadly refers to a biologically inactivesubstance used in combination with the active agents of the formulation.An excipient can be used, for example, as a solubilizing agent, astabilizing agent, a diluent, an inert carrier, a preservative, abinder, a disintegrant, a coating agent, a flavoring agent, or acoloring agent. Preferably, at least one excipient is chosen to provideone or more beneficial physical properties to the formulation, such asincreased stability and/or solubility of the active agent(s). Abenzo[b]azepine TLR agonist as described herein is the primary activeagent in the formulations of the present invention. However, abenzo[b]azepine TLR agonist may be formulated with other active agents,e.g., other TLR agonists, anti-cancer agents or anti-viral agents, asdescribed herein.

A “pharmaceutically acceptable” excipient is one that has been approvedby a state or federal regulatory agency for use in animals, andpreferably for use in humans, or is listed in the U.S. Pharmacopia, theEuropean Pharmacopia or another generally recognized pharmacopia for usein animals, and preferably for use in humans.

Examples of excipients include certain inert proteins such as albumins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asaspartic acid (which may alternatively be referred to as aspartate),glutamic acid (which may alternatively be referred to as glutamate),lysine, arginine, glycine, and histidine; fatty acids and phospholipidssuch as alkyl sulfonates and caprylate; surfactants such as sodiumdodecyl sulphate and polysorbate; nonionic surfactants such as such asTWEEN®, PLURONICS®, or polyethylene glycol (PEG); carbohydrates such asglucose, sucrose, mannose, maltose, trehalose, and dextrins, includingcyclodextrins; polyols such as mannitol and sorbitol; chelating agentssuch as EDTA; and salt-forming counter-ions such as sodium.

The formulations of the present invention preferably contain acyclodextrin which increases the aqueous solubility of the TLR agonist.Cyclodextrins are crystalline, nonhygroscopic cyclic oligomers ofα-D-glucopyranose. As a result of a lack of rotation about the bondsconnecting the glucopyranose units, the cyclodextrins are notcylindrical, but toroidal in shape. Because of this restricted rotationthey have a rigid structure with a central cavity whose size variesaccording to the number of glucopyranose units in the molecule. Thethree most common cyclodextrins are α-cyclodextrin, β-cyclodextrin andγ-cyclodextrin, which consist of six, seven, or eight glucopyranoseunits, respectively. Due to the arrangement of hydroxyl groups withinthe cyclodextrin molecule and the shape of the molecule, the internalsurface of the cavity is hydrophobic, while the outside surface ishydrophilic. The primary hydroxyl groups are located on the narrower(inner) side of the toroidal molecule, while the secondary hydroxylgroups are located on the wider (outer) edge. This arrangement permitsthe cyclodextrins to accommodate a wide variety of small hydrophobicmolecules within the hydrophobic cavity by forming an inclusion complex.

Suitable cyclodextrins for use in the formulations of the invention areknown in the art. For example, TRAPPSOL™ and other cyclodextrins aremade by CTD, Inc. (High Springs, Fla.), and CAPTISOL® (sulfobutyletherβ-cyclodextrin) is present in commercially available injectables such asABILIFY IM™, GEODON, and VFEND IV. Preferably, CAPTISOL® is used in theformulations of the present invention.

In certain embodiments, the formulation comprises 1-30%, 2-12%, 5-15%,15-20%, 20-25%, or 25-30% w/v of a cyclodextrin, preferably aβ-cyclodextrin, and most preferably sulfobutylether β-cyclodextrin. Inother embodiments, the formulation comprises 2%, 4%, 6%, 8%, 10%, 12%,or 15% w/v of a cyclodextrin, preferably a β-cyclodextrin, and mostpreferably sulfobutylether β-cyclodextrin. In other embodiments, theformulation comprises 20%, 25%, or 30% w/v of a cyclodextrin, preferablya β-cyclodextrin, and most preferably sulfobutylether β-cyclodextrin. Ina particular embodiment, the formulation comprises 5%, 15%, 25%, or 30%of a cyclodextrin, preferably a β-cyclodextrin, and most preferablysulfobutylether β-cyclodextrin.

Although cyclodextrins are the preferred solubilizing agents, otherwater-solubilizing agents may be used. Examples of other such agentsinclude Poloxamer, Povidone K17, Povidone K12, Tween 80, ethanol,Cremophor/ethanol, polyethylene glycol 300, polyethylene glycol 400, andpropylene glycol. In preferred embodiments, the formulations of theinvention contain less than 10% v/v of such agents. In certainembodiments, oil-based solubilizing agents such as lipiodol and peanutoil, are used.

The formulations of the present invention may also containpharmaceutically acceptable salts, buffering agents, or preservatives.Examples of such salts include those prepared from the following acids:hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,salicylic, citric, boric, formic, malonic, succinic, and the like. Suchsalts can also be prepared as alkaline metal or alkaline earth salts,such as sodium, potassium or calcium salts. Examples of buffering agentsinclude phosphate, citrate, acetate, and 2-(N-morpholino)ethanesulfonicacid (MES). Examples of preservatives include antioxidants such asvitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium; theamino acids cysteine and methionine; citric acid and sodium citrate; andsynthetic preservatives such as thimerosal, and alkyl parabens,including for example, methyl paraben and propyl paraben. Otherpreservatives include octadecyldimethylbenzyl ammonium chloride,hexamethonium chloride, benzalkonium chloride, benzethonium chloride,phenol, butyl or benzyl alcohol, chlorobutanol, catechol, resorcinol,cyclohexanol, 3-pentanol, and m-cresol.

In certain embodiments, the formulations of the invention may beprepared as a liquid or in a solid form such as a powder, tablet, pillor capsule. Liquid formulations may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. In one embodiment, the formulation is an aqueous solution. Inanother embodiment, the final formulation is lyophilized. In otherembodiments, the formulation comprises a colloidal drug delivery system.Such drug delivery systems include, for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules.

In one embodiment, the benzo[b]azepine TLR agonist formulation is aliquid or lyophilized formulation suitable for injection in a mammal,preferably a human. In one embodiment, the formulation is sterile. Inanother embodiment, the formulation is a sterile lyophilized formulationwhich is suitable for injection upon reconstitution with an amount of anaqueous carrier. In one embodiment, the liquid or lyophilizedformulation is prepared as a unit dosage form as described below. Theformulations may or may not contain an added preservative.

In certain embodiments, the formulations further comprise one or moreadjuvants. Examples of suitable adjuvants include potentiators of theimmune response such as microbial derivatives (e.g., bacterial products,toxins such as cholera toxin and heat labile toxin from E. coli, lipids,lipoproteins, nucleic acids, peptidoglycans, carbohydrates, peptides),cells, cytokines, (e.g., dendritic cells, IL-12, and GM-CSF), hormones,and small molecules. Adjuvants contemplated include, but are not limitedto, oil-based adjuvants (e.g., Freund's adjuvant), CpG oligonucleotides,aluminum salt adjuvants, calcium salt adjuvants, emulsions andsurfactant-based formulations (e.g., MF59, ASO2, montanide, ISA-51,ISA-720, and QA21).

According to certain embodiments, the benzo[b]azepine TLR agonist isformulated at a concentration of from about 0.5 to about 50 mg/ml. Insome embodiments, the benzo[b]azepine TLR agonist is formulated at aconcentration of from about 1 mg/ml to about 5 mg/ml, from about 1 mg/mlto about 10 mg/ml, from about 1 mg/ml to about 20 mg/ml, or from about 1mg/ml to about 30 mg/ml. In other embodiments, the benzo[b]azepine TLRagonist is formulated at a concentration of from about 0.5 mg/ml toabout 1 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, or from about 0.5mg/ml to about 5 mg/ml. In certain embodiments, the benzo[b]azepine TLRagonist is formulated at a concentration of between 0.5 and 10 mg/ml,between 0.5 and 5 mg/ml, or between 1 and 5 mg/ml. In other embodiments,the benzo[b]azepine TLR agonist is formulated at a concentration ofbetween 10-20 mg/ml, 20-30 mg/ml, or between 30-50 mg/ml. In specificembodiments, the benzo[b]azepine TLR agonist is formulated at aconcentration of about 1 mg/ml, about 2 mg/ml, about 4 mg/ml, about 5mg/ml, about 6 mg/ml, about 8 mg/ml, about 10 mg/ml, about 15 mg/ml,about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, or about 40 mg/ml.

In certain embodiments, the pH of the benzo[b]azepine TLR agonistformulation is acidic (meaning less than 7). Preferably, the pH of thebenzo[b]azepine TLR agonist formulation is from about 5.0 to about 7,preferably from about 5.5 to about 6.5. In a particular preferredembodiment, the pH is 6.5.

In certain embodiments, the benzo[b]azepine TLR agonist is formulated inpolyethylene glycol or propylene glycol at a concentration of at least 5mg/ml, at least 8 mg/ml, or at least 9 mg/ml. In accordance with oneaspect of these embodiments, the polyethylene glycol or propylene glycolis present in solution at between 10-20% v/v. In accordance with anotheraspect of these embodiments, both polyethylene glycol and propyleneglycol are present in solution, each at 7.5%, 10%, or 15% v/v.

In certain embodiments, the benzo[b]azepine TLR agonist is formulated ina cyclodextrin, preferably β-cyclodextrin, and most preferablysulfobutylether β-cyclodextrin at a concentration of from about 0.5mg/ml to about 50 mg/ml. In accordance with one aspect of theseembodiments, the cyclodextrin, preferably a β-cyclodextrin, is about1-30% w/v. In specific aspects of these embodiments, the cyclodextrin,preferably a β-cyclodextrin, is about 1% w/v, about 2% w/v, about 5%w/v, about 10% w/v, about 15% w/v, about 20% w/v, about 25% w/v, orabout 30% w/v.

In embodiments where the formulation is an emulsion, suitableemulsifiers or emulsifying agents include any pharmaceuticallyacceptable emulsifier, preferably phospholipids extracted from egg yolkor soy bean, synthetic phosphatidyl cholines or purified phosphatidylcholines from vegetable origin. Hydrogenated derivatives can also beused, such as phosphatidylcholine hydrogenated (egg) andphosphatidylcholine hydrogenated (soya). Emulsifiers may also benon-ionic surfactants such as poloxamers (for example Poloxamer 188 and407), poloxamines, polyoxyethylene stearates, polyoxyethylene sorbitanfatty acid esters or sorbitan fatty acid esters. Ionic surfactants mayalso be used such as cholic acid and deoxycholic acid or surface activederivatives or salts thereof. The emulsifier can also be a mixture ofone or more of the above ingredients. The emulsion may additionallycontain other ingredients such as buffers, stabilizers and other lipids.

The formulations of the present invention can optionally be prepared asunit dosage forms. “Unit dosage form” refers to physically discreteunits suitable for the intended use, i.e., as a single administration tothe subject to be treated. Each unit contains a predetermined quantityof the active agent(s) formulated with the appropriate pharmaceuticallyacceptable excipient(s). For example, a unit dosage per vial may containa certain volume, such as 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8ml, 9 ml, 10 ml, 15 ml, or 20 ml, having a particular concentration ofthe active agent. A dosage unit may comprise a single active agent,i.e., a benzo[b]azepine TLR agonist as described herein, its derivativesand analogs, or mixtures thereof with other active agents for use incombination therapies. In preferred embodiments, the unit dosage formcomprises about 15 mg/ml to about 40 mg/ml of a benzo[b]azepine TLRagonist. The formulations are optionally contained in unit-dose ormulti-dose containers, for example, in sealed ampules or vials, and maybe in a lyophilized condition. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsaccording to art-recognized methods. Examples of unit dosage formsinclude, but are not limited to: tablets; caplets; capsules, such assoft elastic gelatin capsules; cachets; troches; lozenges; dispersions;suppositories; ointments; cataplasms (poultices); pastes; powders;dressings; creams; plasters; solutions; patches; aerosols (e.g., nasalsprays or inhalers); gels; liquid dosage forms suitable for oral ormucosal administration to a patient, including suspensions (e.g.,aqueous or non aqueous liquid suspensions, oil in water emulsions, or awater in oil liquid emulsions), solutions, and elixirs; liquid dosageforms suitable for subcutaneous administration to a subject; and sterilesolids (e.g., crystalline or amorphous solids) that can be reconstitutedto provide liquid dosage forms suitable for subcutaneous administrationto a subject.

Sustained-release preparations may also be prepared. Sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the benzo[b]azepine TLR agonist, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof such matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release molecules for shorter timeperiods.

In certain preferred embodiments, a benzo[b]azepine TLR agonistformulation of the invention comprises 1-30% w/v of a β-cyclodextrin,most preferably sulfobutylether β-cyclodextrim, and one or more of thefollowing benzo[b]azepine TLR agonists at a concentration of about 2mg/ml, about 5 mg/ml, about 10 mg/ml, or about 15 mg/ml, in a formsuitable for injection to a mammal, preferably a human: (1E, 4E)-ethyl2-amino-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxylate; (1E,4E)-2-amino-N,N-bis(2-methoxyethyl)-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-N,N-diethyl-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-8-(perfluoroethyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-N-ethyl-8-(perfluoroethyl)-3H-benzo[b]azepine-4-carboxamide;(1E,4E)-2-amino-8-(perfluoroethyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide;(1E, 4E)-ethyl2-amino-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxylate;(1E, 4E)-ethyl2-amino-8-(4-(methoxycarbonyl)phenyl)-3H-benzo[b]azepine-4-carboxylate;(1E, 4E)-ethyl2-amino-8-(4-(methylcarbamoyl)phenyl)-3H-benzo[b]azepine-4-carboxylate;(1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamide;and pharmaceutically acceptable salts thereof.

Additional information with regard to the methods of making thecompositions and formulations and the ingredients comprising thecompositions and formulations in accordance with the present inventioncan be found in standard references in the field, such as for example,“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.

5.1.1 Stability

The formulations of the present invention provide for the chemicalstability of the formulated benzo[b]azepine TLR agonist. “Stability” and“stable” in the context of the present invention refers to theresistance of the benzo[b]azepine TLR agonist to chemical degradationunder given manufacturing, preparation, transportation and storageconditions. The “stable” formulations of the invention also preferablyretain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the biologicalactivity of a standard or reference preparation of the benzo[b]azepineTLR agonist under given manufacturing, preparation, transportation,and/or storage conditions. Biological activity refers to the ability ofthe benzo[b]azepine TLR agonist to activate TLR signaling, preferablyTLR7 and/or TLR8 signaling, and most preferably TLR8 signaling. In thiscontext, biological activity can be measured using any art-recognizedmethod for detecting TLR signaling. For example, such methods includeassays for the detection of TLR-dependent intracellular signaltransduction molecules, such as nuclear factor kappa beta (NFkB). Suchassays include, e.g., reporter gene assays conducted in cells thatstably express one or more TLR genes, preferably TLR 7 and/or TLR8genes. Other methods include assays for the detection of cytokines whichare released by TLR-containing cells of the immune system when TLRsignaling is activated. For example, assays for the detection of tumornecrosis factor alpha (TNFa) or interferon alpha (IFNα) in thesupernatant of cultured cells are known to the skilled person and arecommercially available (see e.g., R&D Systems, Minneapolis, Minn.).

The “stable” formulations of the invention also preferably retain atleast 90%, 95%, 98%, 99%, or 99.5% of a starting or reference amount ofthe benzo[b]azepine TLR agonist under given manufacturing, preparation,transportation, and/or storage conditions. The amount of benzo[b]azepineTLR agonist can be determined using any art-recognized method. Forexample, benzo[b]azepine TLR agonist concentration can be determinedusing routine methods such as UV-Vis spectrophotometry and high pressureliquid chromatography (HPLC).

In certain embodiments, the present formulations are stable attemperatures ranging from about 20 to 30° C. for at least 1 week, atleast 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, atleast 6 weeks, or at least 7 weeks. In other embodiments, theformulations are stable at temperatures ranging from about 20 to 30° C.for at least 1 month, at least 2 months, at least 3 months, at least 4months, at least 5 months, at least 6 months, at least 7 months, atleast 8 months, at least 9 months, at least 10 months, at least 11months, or at least 12 months. In one embodiment, the formulation isstable for at least 2 months at 20-25° C.

In other embodiments, the present formulations are stable attemperatures ranging from about 2 to 8° C. for at least 1 month, atleast 2 months, at least 4 months, at least 6 months, at least 8 months,at least 10 months, at least 12 months, at least 14 months, at least 16months, at least 18 months, at least 20 months, at least 22 months, orat least 24 months. In one embodiment, the formulation is stable for atleast 2 months at 2 to 8° C.

In other embodiments, the present formulations are stable attemperatures of about −20° C. for at least 1 month, at least 2 months,at least 4 months, at least 6 months, at least 8 months, at least 10months, at least 12 months, at least 14 months, at least 16 months, atleast 18 months, at least 20 months, at least 22 months, or at least 24months. In one embodiment, the formulation is stable for at least 6-12months at 20° C.

In a particular embodiment, a benzo[b]azepine TLR agonist formulation ofthe invention is stable at temperatures of about 20-30° C. atconcentrations up to 40 mg/ml for at least 2 weeks, preferably for atleast 4 weeks, and most preferably for at least 8 weeks. In anotherembodiment, the formulation is stable at temperatures from about 2-8° C.at concentrations up to 40 mg/ml for at least 4 weeks, preferably for atleast 8 weeks, and most preferably for at least 12 weeks. In a anotherembodiment, the formulation is stable at temperatures of about −20° C.at concentrations up to 40 mg/ml for at least 8 weeks, preferably for atleast 12 weeks, and most preferably for at least 16 weeks.

5.2 Methods of Use

The benzo[b]azepine TLR agonist formulations of the present inventionare useful in methods for the treatment of cancer or infectiousdiseases. Preferably, the benzo[b]azepine TLR agonist formulations areused in combination with one or more additional treatment modalities ina regiment for the treatment of cancer. In certain embodiments, thecancer is a solid tumor. In one embodiment, the cancer is selected fromthe group consisting of ovarian cancer, breast cancer, head and neckcancer, renal cancer, bladder cancer, hepatocellular cancer, andlymphoma. In a particular embodiment, the cancer is a lymphoma. In oneembodiment, the lymphoma is non-Hodgkins lymphoma. The benzo[b]azepineTLR agonist formulations of the present invention are also useful inother methods including methods for the treatment of certain skinconditions or diseases, such as atopic dermatitis, methods for thetreatment of infectious diseases, preferably viral diseases, and for useas adjuvants in vaccines formulated for use in cancer therapy or for thetreatment or prevention of infectious diseases, preferably viraldiseases. In one embodiment, the infectious disease is a viral diseaseand the virus is a hepatitis virus, preferably hepatitis C virus (HCV orHepC). The benzo[b]azepine TLR agonist formulations of the invention canbe used either alone or in combination with one or more other treatmentmodalities as described in Section 5.3.1.

5.2.1 Combination Therapy

Combination therapy encompasses, in addition to the administration of abenzo[b]azepine TLR agonist formulation of the invention, the adjunctiveuse of one or more modalities that aid in the prevention or treatment ofcancer. Such modalities include, but are not limited to,chemotherapeutic agents, immunotherapeutics, anti-angiogenic agents,cytokines, hormones, antibodies, polynucleotides, radiation andphotodynamic therapeutic agents. In specific embodiments, combinationtherapy can be used to prevent the recurrence of cancer, inhibitmetastasis, or inhibit the growth and/or spread of cancer or metastasis.As used herein, “in combination with” means that the benzo[b]azepine TLRagonist formulation of the invention is administered as part of atreatment regimen that comprises one or more additional treatmentmodalities as described in more detail in the following sections.

In certain embodiments, the benzo[b]azepine TLR agonist is administeredprior to, concurrently with, or subsequent to the administration of theone or more other modalities. In one embodiment the benzo[b]azepine TLRagonist is formulated with one or more other modalities. In anotherembodiment, the one or more other modalities is administered in aseparate pharmaceutical composition. In accordance with this embodiment,the one or more other modalities may be administered to a subject by thesame or different routes of administration as those used to administerthe benzo[b]azepine TLR agonist.

5.2.1.1 Combination with Anti-cancer Agents

In certain embodiments, the formulation comprising a benzo[b]azepine TLRagonist of the invention is administered in combination with one or moreanti-cancer agents, preferably a chemotherapeutic agent. Suchchemotherapeutic agents include, but are not limited to, the followinggroups of compounds: cytotoxic antibiotics, antimetabolities,anti-mitotic agents, alkylating agents, platinum compounds, arseniccompounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues,plant alkaloids, and toxins; and synthetic derivatives thereof. Thefollowing are non-limiting examples of particular compounds within thesegroups. Alkylating agents include nitrogen mustards such ascyclophosphamide, ifosfamide, trofosfamide, and chlorambucil;nitrosoureas such as carmustine (BCNU) and lomustine (CCNU);alkylsulphonates such as busulfan and treosulfan; and triazenes such asdacarbazine. Platinum containing compounds include cisplatin,carboplatin, aroplatin, and oxaliplatin. Plant alkaloids include vincaalkaloids such as vincristine, vinblastine, vindesine, and vinorelbine;and taxoids such as paclitaxel and docetaxol. DNA topoisomeraseinhibitors include epipodophyllins such as etoposide, teniposide,topotecan, 9-aminocamptothecin, camptothecin, and crisnatol; andmitomycins such as mitomycin C. Anti-folates include DHFR inhibitorssuch as methotrexate and trimetrexate; IMP dehydrogenase inhibitors suchas mycophenolic acid, tiazofurin, ribavirin, hydroxyurea and EICAR; andribonuclotide reductase inhibitors such as deferoxamine. Pyrimidineanalogs include uracil analogs such as 5-fluorouracil, floxuridine,doxifluridine, and ratitrexed; and cytosine analogs such as cytarabine(ara C), cytosine arabinoside, and fludarabine. Purine analogs includemercaptopurine and thioguanine. DNA antimetabolites include 3-HP,2′-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate,ara-C, 5-aza-2′-deoxycytidine, beta-TGDR, cyclocytidine, guanazole,inosine glycodialdehyde, macebecin II, and pyrazoloimidazole.Antimitotic agents include allocolchicine, halichondrin B, colchicine,colchicine derivative, dolstatin 10, maytansine, rhizoxin,thiocolchicine, and trityl cysteine.

Other examples of chemotherapeutic agents for use with thebenzo[b]azepine TLR agonist formulations of the invention includeisoprenylation inhibitors; dopaminergic neurotoxins such as1-methyl-4-phenylpyridinium ion; cell cycle inhibitors such asstaurosporine; actinomycins such as actinomycin D and dactinomycin;bleomycins such as bleomycin A2, bleomycin B2, and peplomycin;anthracyclines such as daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, and mitoxantrone; MDRinhibitors such as verapamil; and Ca²⁺ ATPase inhibitors such asthapsigargin.

Compositions comprising one or more chemotherapeutic agents (e.g., FLAG,CHOP) are also contemplated for use in combination with thebenzo[b]azepine TLR agonist formulations of the invention. FLAGcomprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOPcomprises cyclophosphamide, vincristine, doxorubicin, and prednisone.Each of the foregoing lists is illustrative, and is not intended to belimiting.

In one embodiment, the benzo[b]azepine TLR agonist formulation of theinvention is administered in combination with one or more of thefollowing: IFNα, IL-2, Dacarbazine (Bayer), Temozolomide (Schering),Tamoxifen (AZ), Carmustine (BMS), Melphalan (GSK), Procarbazine(Sigma-Tau), Vinblastine, carboplatin, cisplatin, taxol,cyclophosphamide, doxorubin, Rituxan (Genentech/Roche), Herceptin(Genentech/Roche), Gleevec, Iressa (AZ), Avastin (Genentech/Roche), orTarceva (Genentech/Roche).

In another embodiment, the benzo[b]azepine TLR agonist formulation ofthe invention is administered in combination with one or more of thefollowing: an enediyne such as calicheamicin and esperamicin;duocarmycin, methotrexate, doxorubicin, melphalan, chlorambucil, Ara-C,vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, and5-fluorouracil.

Suitable toxins and chemotherapeutic agents that can be used incombination with the benzo[b]azepine TLR agonist formulations of thisinvention are described in Remington's Pharmaceutical Sciences, 19th Ed.(Mack Publishing Co. 1995), and in Goodman and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985). Other suitable toxins and/or chemotherapeutic agents are known tothose of skill in the art.

Further examples of anti-cancer agents that can be used in combinationwith the benzo[b]azepine TLR agonist formulations of this inventioninclude without limitation the following: acivicin; aclarubicin;acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other anti-cancer agents that can be used include, but are not limitedto: 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustardanti-cancer agent; mycaperoxide B; mycobacterial cell wall extract;myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides;onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxelanalogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

5.2.1.2 Combination with Radiation Therapy

In another embodiment, the benzo[b]azepine TLR agonist formulations ofthe invention are administered in conjunction with a regimen ofradiation therapy for the treatment of cancer. The methods encompassregimens comprising external-beam radiation therapy, interstitialimplantation of radioisotopes (I-125, palladium, iridium), radioisotopessuch as strontium-89, thoracic radiation therapy, intraperitoneal P-32radiation therapy, and/or total abdominal and pelvic radiation therapy.Any suitable cytotoxic radionuclide or therapeutic isotope may be usedin the regimen of radiation therapy. In certain embodiments, the isotopeis an alpha-emitting isotope such as ²²⁵Ac, ²²⁴Ac, ²¹¹At, ²¹²Bi, ²¹³Bi,²¹²Pb, ²²⁴Ra, or ²²³Ra. In other embodiments, the cytotoxic radionuclideis a beta-emitting isotope such as ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹⁷⁷Lu,¹⁵³Sm, ¹⁶⁶Ho, or ⁶⁴Cu. In some embodiments, cytotoxic radionuclide is anisotope that emits Auger and low energy electrons such as ¹²⁵I, ¹²³I or⁷⁷Br. In other embodiments the isotope is ¹⁹⁸Au, ³²P, and the like.

In certain embodiments, the amount of the radionuclide administered tothe subject is between about 0.001 mCi/kg and about 10 mCi/kg. In someembodiments, the amount of the radionuclide administered to the subjectis between about 0.1 mCi/kg and about 1.0 mCi/kg. In other embodiments,the amount of the radionuclide administered to the subject is betweenabout 0.005 mCi/kg and 0.1 mCi/kg.

5.2.1.3 Combination with Therapeutic Antibodies

In another embodiment, a benzo[b]azepine TLR agonist formulation of theinvention is administered in combination with one or moreimmunotherapeutic agents, such as an antibody or a vaccines. In someembodiments, the antibodies have in vivo therapeutic and/or prophylacticuses against cancer. In some embodiments, the antibodies can be used fortreatment and/or prevention of infectious disease.

Non-limiting examples of therapeutic and prophylactic antibodies thatcan be used in combination with a benzo[b]azepine TLR agonistformulation of the invention include MDX-010 (Medarex, NJ) which is ahumanized anti-CTLA-4 antibody currently in clinic for the treatment ofprostate cancer; SYNAGIS® (MedImmune, MD) which is a humanizedanti-respiratory syncytial virus (RSV) monoclonal antibody for thetreatment of RSV infection; and HERCEPTIN® (Trastuzumab) (Genentech, CA)which is a humanized anti-HER2 monoclonal antibody for the treatment ofmetastatic breast cancer. Other examples are humanized anti-CD18 F(ab′)₂(Genentech); CDP860 which is a humanized anti-CD18 F(ab′)₂ (Celltech,UK); PRO542 which is an anti-HIV gp120 antibody fused with CD4(Progenics/Genzyme Transgenics); Ostavir which is a human anti-HepatitisB virus antibody (Protein Design Lab/Novartis); PROTOVIR™ which is ahumanized anti-CMV IgG1 antibody (Protein Design Lab/Novartis); MAK-195(SEGARD) which is a murine anti-TNF-α F(ab′)₂ (Knoll Pharma/BASF); IC14which is an anti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgG1antibody (Genentech); OVAREX™ which is a murine anti-CA 125 antibody(Altarex); PANOREX™ which is a murine anti-17-IA cell surface antigenIgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murineanti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™which is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti-CD52IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics);Smart ID10 which is a humanized anti-HLA antibody (Protein Design Lab);ONCOLYM™ (Lym-1) is a radiolabelled murine anti-HLA DIAGNOSTIC REAGENTantibody (Techniclone); ABX-IL8 is a human anti-IL8 antibody (Abgenix);anti-CD11a is a humanized IgG1 antibody (Genentech/Xoma); ICM3 is ahumanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatizedanti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is a radiolabelledmurine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanizedanti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF);CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is aprimatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is ahumanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A isa humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ is ahumanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanizedanti-VLA-4 IgG antibody (Elan); MDX-33 is a human anti-CD64 (FcγR)antibody (Medarex/Centeon); SCH55700 is a humanized anti-IL-5 IgG4antibody (Celltech/Schering); SB-240563 and SB-240683 are humanizedanti-IL-5 and IL-4 antibodies, respectively, (SmithKline Beecham);rhuMab-E25 is a humanized anti-IgE IgG1 antibody(Genentech/Norvartis/Tanox Biosystems); ABX-CBL is a murine anti CD-147IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(Medimmune/Bio Transplant); Orthoclone/OKT3 is a murine anti-CD3 IgG2aantibody (ortho Biotech); SIMULECT™ is a chimeric anti-CD25 IgG1antibody (Novartis Pharm); LDP-01 is a humanized anti-β₂-integrin IgGantibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab′)₂(Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-β₂ antibody(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VIIantibody (Centocor). The above-listed immunoreactive reagents, as wellas any other immunoreactive reagents, may be administered according toany regimen known to those of skill in the art, including the regimensrecommended by the suppliers of the immunoreactive reagents.

5.2.1.4 Combination with Other Therapeutic Agents

In addition to anti-cancer agents and therapeutic antibodies, thebenzo[b]azepine TLR agonist formulations of the invention can beadministered in combination with other therapeutic agents such asanti-angiogenic agents (e.g., in methods for the treatment of solidtumors and for the treatment and prevention of metastases) andanti-hormonal agents (particularly in methods for the treatment ofhormone-dependent cancers such as breast cancer and prostate cancer).

In one embodiment, a benzo[b]azepine TLR agonist formulation of theinvention is administered in combination with one or moreanti-angiogenic agents. Such agents include, without limitation,angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine ProteaseInhibitor) anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminalproteolytic fragments of fibronectin, 16 kDa proteolytic fragment ofprolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a β-aminoacid peptide corresponding to a fragment of platelet factor-4 (Maione etal., 1990, Cancer Res. 51:2077-2083), a 14-amino acid peptidecorresponding to a fragment of collagen I (Tolma et al., 1993, J. CellBiol. 122:497-511), a 19 amino acid peptide corresponding to a fragmentof Thrombospondin I (Tolsma et al., 1993, J. Cell Biol. 122:497-511), a20-amino acid peptide corresponding to a fragment of SPARC (Sage et al.,1995, J. Cell. Biochem. 57:1329-1334), or any fragments, family members,or variants thereof, including pharmaceutically acceptable saltsthereof.

Other peptides that inhibit angiogenesis and correspond to fragments oflaminin, fibronectin, procollagen, and EGF have also been described(see, e.g., Cao, 1998, Prog Mol Subcell Biol. 20:161-176). Monoclonalantibodies and cyclic pentapeptides, which block certain integrins thatbind RGD proteins (i.e., possess the peptide motif Arg-Gly-Asp), havebeen demonstrated to have anti-vascularization activities (Brooks etal., 1994, Science 264:569-571; Hammes et al., 1996, Nature Medicine2:529-533). Moreover, inhibition of the urokinase plasminogen activatorreceptor by receptor antagonists inhibits angiogenesis, tumor growth andmetastasis (Min et al., 1996, Cancer Res. 56: 2428-33; Crowley et al.,1993, Proc Natl Acad Sci. 90:5021-25).

In another embodiment, a benzo[b]azepine TLR agonist formulation of theinvention is used in association with a hormonal treatment modality.Such treatment modalities include the administration of hormonalantagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene,leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormonebiosynthesis and processing, and steroids (e.g., dexamethasone,retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone,dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen,testosterone, progestins), vitamin A derivatives (e.g., all-transretinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g.,mifepristone, onapristone), and antiandrogens (e.g., cyproteroneacetate).

In another embodiment, a benzo[b]azepine TLR agonist formulation of theinvention is used in association with a treatment modality that utilizespolynucleotide compounds, such as antisense polynucleotides, ribozymes,RNA interference molecules, triple helix polynucleotides and the like.

5.2.1.5 Combination with Immunoregulatory Agents

In certain embodiments, a benzo[b]azepine TLR agonist formulation of theinvention is administered in combination with an immunoregulatory agent.In some embodiments, the benzo[b]azepine TLR agonist is formulated withthe immunoregulatory agent. An “immunoregulatory agent” is a substancethat suppresses, masks, or enhances the immune system of the subject towhom it is administered. Exemplary agents are those that suppresscytokine production, downregulate or suppress self-antigen expression,or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see, U.S. Pat. No. 4,665,077),azathioprine (or cyclophosphamide, if there is an adverse reaction toazathioprine); bromocryptine; glutaraldehyde (which masks the MHCantigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypicantibodies for MHC antigens and MHC fragments; cyclosporin A; steroidssuch as glucocorticosteroids, e.g., prednisone, methylprednisolone, anddexamethasone; cytokine or cytokine receptor antagonists includinganti-interferon-γ, -β, or -α antibodies; anti-tumor necrosis factor-αantibodies; anti-tumor necrosis factor-β antibodies; anti-interleukin-2antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies;heterologous anti-lymphocyte globulin; pan-T antibodies, preferablyanti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3binding domain; streptokinase; TGF-β; streptodornase; FK506; RS-61443;deoxyspergualin; and rapamycin. Examples of cytokines include, but arenot limited to lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are growth hormone such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-α; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoiotin (TPO); nerve growth factors suchas NGF-α; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-α; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CgP (GM-CSP); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-1 I, IL-12, IL-15; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

In certain embodiments, the methods further include administering to thesubject one or more immunomodulatory agents, preferably a cytokine.Preferred cytokines are selected from the group consisting ofinterleukin-1 (IL-1), IL-2, IL-3, IL-12, IL-15, IL-18, G-CSF, GM-CSF,thrombopoietin, and γ interferon.

5.2.1.6 Combination with Compounds that Enhance Monocyte or MacrophageFunction

In certain embodiments, a compound that enhances monocyte or macrophagefunction (e.g., at least about 25%, 50%, 75%, 85%, 90%, 9% or more) canbe used in conjunction with the benzo[b]azepine TLR agonist formulationsof the invention. Such compounds are known in the art and include,without limitation, cytokines such as interleukins (e.g., IL-12), andinterferons (e.g., alpha or gamma interferon).

In certain embodiments, the compound that enhances monocyte ormacrophage function is formulated with the benzo[b]azepine TLR agonistand is thus administered concurrently with the benzo[b]azepine TLRagonist.

In other embodiments, the compound that enhances monocyte or macrophagefunction is administered separately from the benzo[b]azepine TLR agonistand can be administered concurrently (within a period of hours of eachother), during the same course of therapy, or sequentially with thebenzo[b]azepine TLR agonist. In such embodiments, the compound thatenhances monocyte or macrophage function is preferably administered to ahuman subject. In one embodiment, the human subject has a bloodleukocyte, monocyte, neutrophil, lymphocyte, and/or basophil count thatis within the normal range for humans. Normal ranges for human bloodleukocytes (total) is about 3.5-10.5 (10⁹/L). Normal ranges for humanblood neutrophils is about 1.7-7.0 (10⁹/L), monocytes is about 0.3-0.9(10⁹/L), lymphocytes is about 0.9-2.9 (10⁹/L), basophils is about 0-0.3(10⁹/L), and eosinophils is about 0.05-0.5 (10⁹/L). In otherembodiments, the human subject has a blood leukocyte count that is lessthan the normal range for humans, for example at least about 0.01, 0.05,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 (10⁹/L) leukocytes.

5.2.2 Target Cancers

In certain preferred embodiments, the type of cancer that is treated bythe methods of the present invention is ovarian cancer, breast cancer,head and neck cancer, renal cancer, bladder cancer, hepatocellularcancer, or lymphoma. Other types of cancers that can be treated by themethods of the present invention include, but are not limited to humansarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease.

5.2.3 Infectious Diseases and Agents

The benzo[b]azepine TLR agonist formulations of the present inventioncan be used in methods of treating or preventing an infectious diseasein a subject, preferably a human subject. In certain embodiments, themethod is a method for the treatment or prevention of an infectiousdisease caused by a virus, bacteria, fungi, protozoa, helminth, orparasite, including particular strains thereof.

In certain embodiments, a benzo[b]azepine TLR agonist formulation of theinvention is used in a method of treating a viral disease caused byherpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-8), influenzaviruses (Flu A, B), hepatitis viruses (HepA, HepB, HepC, HepE), humanimmunodeficiency viruses (HIV-1, HIV-2), respiratory syncytial viruses,measles viruses, rhinoviruses, adenoviruses, SARS viruses,papillomaviruses, orthopoxviruses, or West Nile viruses. In a preferredembodiment, the benzo[b]azepine TLR agonist formulations of theinvention are used in a method for the treatment of hepatitis,preferably hepatitis C (also referred to as HCV).

In certain embodiments, the benzo[b]azepine TLR agonist formulation isadministered in combination with an anti-viral agent. Antiviral agentsthat can be used include, but are not limited to, non-nucleoside reversetranscriptase inhibitors, nucleoside reverse transcriptase inhibitors,protease inhibitors, and fusion inhibitors. In one embodiment, theantiviral agent is selected from the group consisting of amantadine,oseltamivir phosphate, rimantadine, and zanamivir. In one embodiment,the antiviral agent is a non-nucleoside reverse transcriptase inhibitorselected from the group consisting of delavirdine, efavirenz, andnevirapine. In one embodiment, the antiviral agent is a nucleosidereverse transcriptase inhibitor selected from the group consisting ofabacavir, didanosine, emtricitabine, emtricitabine, lamivudine,stavudine, tenofovir DF, zalcitabine, and zidovudine. In one embodiment,the antiviral agent is a protease inhibitor selected from the groupconsisting of amprenavir, atazanavir, fosamprenav, indinavir, lopinavir,nelfinavir, ritonavir, and saquinavir. In one embodiment, the antiviralagent is a fusion inhibitor such as enfuvirtide.

Additional, non-limiting examples of antiviral agents for use in themethods of the invention include the following: rifampicin, nucleosidereverse transcriptase inhibitors (e.g., AZT, ddI, ddC, 3TC, d4T),non-nucleoside reverse transcriptase inhibitors (e.g., delavirdineefavirenz, nevirapine), protease inhibitors (e.g., aprenavir, indinavir,ritonavir, and saquinavir), idoxuridine, cidofovir, acyclovir,ganciclovir, zanamivir, amantadine, and palivizumab. Other examples ofanti-viral agents include but are not limited to acemannan; acyclovir;acyclovir sodium; adefovir; alovudine; alvircept sudotox; amantadinehydrochloride (SYMMETREL™); aranotin; arildone; atevirdine mesylate;avridine; cidofovir; cipamfylline; cytarabine hydrochloride; delavirdinemesylate; desciclovir; didanosine; disoxaril; edoxudine; enviradene;enviroxime; famciclovir; famotine hydrochloride; fiacitabine;fialuridine; fosarilate; foscamet sodium; fosfonet sodium; ganciclovir;ganciclovir sodium; idoxuridine; kethoxal; lamivudine; lobucavir;memotine hydrochloride; methisazone; nevirapine; oseltamivir phosphate(TAMIFLU™); penciclovir; pirodavir; ribavirin; rimantadine hydrochloride(FLUMADINE™); saquinavir mesylate; somantadine hydrochloride;sorivudine; statolon; stavudine; tilorone hydrochloride; trifluridine;valacyclovir hydrochloride; vidarabine; vidarabine phosphate; vidarabinesodium phosphate; viroxime; zalcitabine; zanamivir (RELENZA™);zidovudine; and zinviroxime.

Other examples of viruses encompassed by the methods of the inventioninclude, without limitation, the following viruses: Retroviridae (e.g.human immunodeficiency viruses, such as HIV-1, also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae(e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g. ebola-likeviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2), varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the etiological agents of Spongiform encephalopathies, the agentof delta hepatitis, thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class 1, internallytransmitted; class 2, parenterally transmitted, i.e., Hepatitis C);Norwalk and related viruses, and astroviruses.

In other embodiments, a benzo[b]azepine TLR agonist formulation of theinvention is used in a method of treating a disease caused by abacterium. Non-limiting examples of bacteria encompassed by the methodsof the invention include Mycobacteria, Streptococcus, Staphylococcus,Pseudomonas, Salmonella, Neisseria, and Listeria. Other examples ofbacteria contemplated include, but are not limited to, Gram positivebacteria (e.g., Listeria, Bacillus such as Bacillus anthracis,Erysipelothrix species), Gram negative bacteria (e.g., Bartonella,Brucella, Campylobacter, Enterobacter, Escherichia, Francisella,Hemophilus, Klebsiella, Morganella, Proteus, Providencia, Pseudomonas,Salmonella, Serratia, Shigella, Vibrio, and Yersinia species),spirochete bacteria (e.g., Borrelia species including Borreliaburgdorferi that causes Lyme disease), anaerobic bacteria (e.g.,Actinomyces and Clostridium species), Gram positive and negative coccalbacteria, Enterococcus species, Streptococcus species, Pneumococcusspecies, Staphylococcus species, Neisseria species. Additionalnon-limiting examples of specific infectious bacteria includeHelicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria avium, M. intracellulare, M. kansaii, M. gordonae, M.africanum, Staphylococcus aureus, Neisseria meningitidis, Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Fusobacterium nucleatum, Streptobacillus moniliformis,Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, andActinomyces israelli.

In certain embodiments, a benzo[b]azepine TLR agonist formulation of thepresent invention is administered in combination with an anti-bacterialagent. Non-limiting examples of anti-bacterial agents include proteins,polypeptides, peptides, fusion proteins, antibodies, nucleic acidmolecules, organic molecules, inorganic molecules, and small moleculesthat inhibit and/or reduce a bacterial infection, inhibit and/or reducethe replication of bacteria, or inhibit and/or reduce the spread ofbacteria to other cells or subjects. Specific examples of anti-bacterialagents include, but are not limited to, antibiotics such as penicillin,cephalosporin, imipenem, axtreonam, vancomycin, cycloserine, bacitracin,chloramphenicol, erythromycin, clindamycin, tetracycline, streptomycin,tobramycin, gentamicin, amikacin, kanamycin, neomycin, spectinomycin,trimethoprim, norfloxacin, rifampin, polymyxin, amphotericin B,nystatin, ketocanazole, isoniazid, metronidazole, and pentamidine.

In certain embodiments a benzo[b]azepine TLR agonist formulation of theinvention is administered in combination with an anti-fungal agent.Specific examples of anti-fungal agents include, but are not limited to,azole drugs (e.g., miconazole, ketoconazole (NIZORAL®), caspofunginacetate (CANCIDAS®), imidazole, triazoles (e.g., fluconazole(DIFLUCAN®), and itraconazole (SPORANOX®)), polyene (e.g., nystatin,amphotericin B (FUNGIZONE®), amphotericin B lipid complex(“ABLC”)(ABELCET®), amphotericin B colloidal dispersion(“ABCD”)(AMPHOTEC®), liposomal amphotericin B (AMBISONE®)), potassiumiodide (KI), pyrimidine (e.g., flucytosine (ANCOBON®)), and voriconazole(VFEND®).

5.3 Administration and Dosing

The benzo[b]azepine TLR agonists of the invention are preferablyformulated for injection, most preferably subcutaneous injection. Incertain embodiments, the benzo[b]azepine TLR agonists of the inventionare formulated for administration by an intradermal, a transdermal, asubcutaneous, or an intramuscular route. In one embodiment, thebenzo[b]azepine TLR agonists are formulated for intravenousadministration. However, the benzo[b]azepine TLR agonists may beformulated for any suitable route of administration, including, by wayof example, nasal (e.g., via an aerosol), buccal (e.g., sub-lingual),topical (i.e., both skin and mucosal surfaces, including airwaysurfaces), intrathecal, intra-articular, intraplural, intracerebral,intra-arterial, intraperitoneal, oral, intralymphatic, intranasal,rectal or vaginal administration, by perfusion through a regionalcatheter, or by direct intralesional injection.

The formulations of the present invention contain an amount of abenzo[b]azepine TLR agonist that is effective for the intended use.Particular dosages are also selected based on a number of other factorsincluding the age, sex, species and condition of the patient. Effectiveamounts can also be extrapolated from dose-response curves derived fromin vitro test systems or from animal models.

In certain embodiments, the dose of benzo[b]azepine TLR agonist ismeasured in units of mg/kg of body weight. In other embodiments, thedose is measured in units of mg/kg of lean body weight (i.e., bodyweight minus body fat content). In other embodiments, the dose ismeasured in units of mg/m² of body surface area. In other embodiments,the dose is measured in units of mg per dose administered to a patient.Any measurement of dose can be used in conjunction with the compositionsand methods of the invention and dosage units can be converted by meansstandard in the art.

Examples of dosing regimens that can be used in the methods of theinvention include, but are not limited to, daily, three times weekly(intermittent), weekly, or every 14 days. In certain embodiments, dosingregimens include, but are not limited to, monthly dosing or dosing every6-8 weeks. In a preferred embodiment, a benzo[b]azepine TLR agonistformulation of the present invention is administered by subcutaneousinjection weekly or biweekly in combination with a suitable treatmentmodality for the treatment of cancer or infectious disease in a subject,preferably a human subject. Suitable treatment modalities are describedin Section 5.3 and its subsections.

Exemplary doses of a benzo[b]azepine TLR agonist include milligramamounts per kilogram of the subject. In one embodiment, the dose is fromabout 0.02 to 10 mg/kg of body weight or about 0.04 to 5 mg/kg of bodyweight. In a specific embodiment, the dosage is about 0.05 mg/kg, about0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, or about 10mg/kg of the subject's body weight.

In certain embodiments of the methods for treating cancer or infectiousdisease, the benzo[b]azepine TLR agonist is administered to the subjectat a dose of from about 0.02 to 10 mg/kg of body weight or about 0.04 to5 mg/kg of body weight of the subject. In particular embodiments, thebenzo[b]azepine TLR agonist is administered at a dose of about 0.05mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg,or about 10 mg/kg of the subject's body weight. In certain furtherembodiments, the benzo[b]azepine TLR agonist formulation is administeredto the subject on a weekly or biweekly basis. In specific embodiments, adaily dose is at least 0.05 mg, 0.50 mg, 1.0 mg, 5.0 mg, 10 mg, 15 mg,20 mg, 30 mg, or at least 50 mg.

Recommended dosages for intradermal, intramuscular, intraperitoneal,subcutaneous, epidural, or intravenous administration are in the rangeof about 0.02 to 10 mg/kg of body weight per day. Suitable doses fortopical administration are in the range of about 0.001 milligram toabout 50 milligrams, depending on the area of administration. Thoseskilled in the art will appreciate that dosages are generally higherand/or frequency of administration greater for initial treatment ascompared with maintenance regimens.

5.3.1 Exemplary Regimens for the Treatment of Cancer

In particular embodiments, the benzo[b]azepine TLR agonist formulationsof the invention are used in combination with an existing treatmentregimen for the treatment of cancer in a subject, preferably a humansubject. In accordance with this embodiment, the benzo[b]azepine TLRagonist formulation can be administered prior to, subsequently, orconcurrently with a suitable anti-cancer agent(s) for the treatment ofcancer. Preferably, the administration of the benzo[b]azepine TLRagonist formulation is coordinated with the dosage and timing of theanti-cancer agent(s) depending on the type of cancer, the subject'shistory and condition, and the particular anti-cancer agent(s) ofchoice.

In one embodiment, the regimen comprises 5-fluorouracil, cisplatin,docetaxel, doxorubicin, HERCEPTIN®, gemcitabine, IL-2, paclitaxel,and/or VP-16 (etoposide) for the treatment of breast cancer. In anotherembodiment, the regimen comprises paclitaxel, docetaxel, mitoxantrone,and/or an androgen receptor antagonist (e.g., flutamide) for thetreatment of prostate cancer. In another embodiment, the regimencomprises fludarabine, cytosine arabinoside, gemtuzumab (MYLOTARG),daunorubicin, methotrexate, vincristine, 6-mercaptopurine, idarubicin,mitoxantrone, etoposide, asparaginase, prednisone and/orcyclophosphamide for the treatment of leukemia. In one embodiment, theregimen comprises dexamethasone for the treatment of myeloma. In oneembodiment, the regimen comprises dacarbazine for the treatment ofmelanoma. In one embodiment, the regimen comprises irinotecan for thetreatment of colorectal cancer. In one embodiment, the regimen comprisespaclitaxel, docetaxel, etoposide and/or cisplatin for the treatment oflung cancer. In one embodiment, the regimen comprises cyclophosphamide,CHOP, etoposide, bleomycin, mitoxantrone and/or cisplatin for thetreatment of non-Hodgkin's lymphoma. In one embodiment, the regimencomprises cisplatin for the treatment of gastric cancer. In oneembodiment, the regimen comprises gemcitabine for the treatment ofpancreatic cancer.

The duration of treatment with the anti-cancer agent may vary accordingto the particular therapeutic agent used. In certain embodiments, theadministration is discontinuous, i.e., daily doses are divided intoseveral partial administrations. According to certain embodiments, themethod of treatment comprises at least one cycle, preferably more thanone cycle, during which a single therapeutic or sequence of therapeuticsis administered. An appropriate period of time for one cycle can bedetermined according to routine methods by the skilled artisan, as wellas the total number of cycles, and the interval between cycles.

In a specific embodiment, the regimen comprises gemcitabine at a doseranging from 100 to 1000 mg/m²/cycle. In another embodiment, the regimencomprises dacarbazine at a dose ranging from 200 to 4000 mg/m²/cycle. Ina preferred embodiment, the dose of dacarbazine ranges from 700 to 1000mg/m²/cycle. In another embodiment, the regimen comprises fludarabine ata dose ranging from 25 to 50 mg/m²/cycle. In another embodiment, theregimen comprises cytosine arabinoside (Ara-C) at a dose ranging from200 to 2000 mg/m²/cycle. In another embodiment, the regimen comprisesdocetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle. In anotherembodiment, the regimen comprises paclitaxel at a dose ranging from 5 to15 mg/kg/cycle. In another embodiment, the regimen comprises cisplatinat a dose ranging from 5 to 20 mg/kg/cycle. In another embodiment, theregimen comprises 5-fluorouracil at a dose ranging from 5 to 20mg/kg/cycle. In another embodiment, the regimen comprises doxorubicin ata dose ranging from 2 to 8 mg/kg/cycle. In another embodiment, theregimen comprises epipodophyllotoxin at a dose ranging from 40 to 160mg/kg/cycle. In another embodiment, the regimen comprisescyclophosphamide at a dose ranging from 50 to 200 mg/kg/cycle. Inanother embodiment, the regimen comprises irinotecan at a dose rangingfrom 50 to 75, 75 to 100, 100 to 125, or 125 to 150 mg/m²/cycle. Inanother embodiment, the regimen comprises vinblastine at a dose rangingfrom 3.7 to 5.4, 5.5 to 7.4, 7.5 to 11, or 11 to 18.5 mg/m²/cycle. Inanother embodiment, the regimen comprises vincristine at a dose rangingfrom 0.7 to 1.4, or 1.5 to 2 mg/m²/cycle. In yet another embodiment, theregimen comprises methotrexate at a dose ranging from 3.3 to 5, 5 to 10,10 to 100, or 100 to 1000 mg/m²/cycle.

In one embodiment, the regimen encompasses the use of a low dose of achemotherapeutic agent. In accordance with this embodiment, initialtreatment of a subject with a benzo[b]azepine TLR agonist formulation ofthe invention increases the sensitivity of a tumor to subsequentchallenge with an anti-cancer agent. Thus, the anti-cancer agent can beadministered to the subject at a dose that is near or below the lowerrange of acceptable dosages for that agent administered alone. In oneembodiment, the regimen comprises the subsequent administration ofdocetaxel at 6 to 60 mg/m²/day or less. In another embodiment, theregimen comprises the subsequent administration of paclitaxel at 10 to135 mg/m²/day or less. In another embodiment, the regimen comprises thesubsequent administration of fludarabine at 2.5 to 25 mg/m²/day or less.In another embodiment, the regimen comprises the subsequentadministration of cytosine arabinoside (Ara-C) at 0.5 to 1.5 g/m²/day orless. In another embodiment, the regimen comprises the subsequentadministration of gemcitabine at from 10 to 100 mg/m²/cycle. In anotherembodiment, the regimen comprises the subsequent administration ofcisplatin, e.g., PLATINOL or PLATINOL-AQ (Bristol Myers), at a doseranging from 5 to 10, 10 to 20, 20 to 40, or 40 to 75 mg/m²/cycle. Inanother embodiment, the regimen comprises the subsequent administrationof cisplatin ranging from 7.5 to 75 mg/m²/cycle. In another embodiment,the regimen comprises the subsequent administration of carboplatin,e.g., PARAPLATIN (Bristol Myers), at a dose ranging from 2 to 4, 4 to 8,8 to 16, 16 to 35, or 35 to 75 mg/m²/cycle. In another embodiment, theregimen comprises the subsequent administration of docetaxel, e.g.,TAXOTERE (Rhone Poulenc Rorer) at a dose ranging from 6 to 10, 10 to 30,or 30 to 60 mg/m²/cycle. In another embodiment, the regimen comprisesthe subsequent administration of paclitaxel, e.g., TAXOL (Bristol MyersSquibb), at a dose ranging from 10 to 20, 20 to 40, 40 to 70, or 70 to135 mg/kg/cycle. In another embodiment, the regimen comprises thesubsequent administration of 5-fluorouracil at a dose ranging from 0.5to 5 mg/kg/cycle. In another embodiment, the regimen comprises thesubsequent administration of doxorubicin, e.g., ADRIAMYCIN (Pharmacia &Upjohn), DOXIL (Alza), RUBEX (Bristol Myers Squibb), at a dose rangingfrom 2 to 4, 4 to 8, 8 to 15, 15 to 30, or 30 to 60 mg/kg/cycle.

The above-described administration schedules are provided forillustrative purposes only and should not be considered limiting.

5.4 Kits

The present invention provides a pharmaceutical pack or kit comprisingone or more containers filled with a liquid or lyophilizedbenzo[b]azepine TLR agonist formulation of the invention. In oneembodiment, the formulation is an aqueous formulation of thebenzo[b]azepine TLR agonist (1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamidecontaining a β-cyclodextrin, preferably sulfobutylether β-cyclodextrin.In one embodiment, the formulation is lyophilized. In preferredembodiments the liquid or lyophilized formulation is sterile. In oneembodiment, the kit comprises a liquid or lyophilized formulation of theinvention, in one or more containers, and one or more other prophylacticor therapeutic agents useful for the treatment of cancer or aninfectious disease. The one or more other prophylactic or therapeuticagents may be in the same container as the benzo[b]azepine TLR agonistor in one or more other containers. Preferably, the benzo[b]azepine TLRagonist is formulated at a concentration of from about 0.5 mg/ml toabout 50 mg/ml, from about 1 mg/ml to about 40 mg/ml, or from about 2mg/ml to about 15 mg/ml, and the formulation is suitable for injection,preferably subcutaneous injection. Preferably, the kit contains thebenzo[b]azepine TLR agonist in unit dosage form. Most preferably, theunit dosage form is in a form suitable to provide a unit dose of about0.02 to 10 mg/kg or about 0.04 to 5 mg/kg of body weight of the subjectto be treated.

In certain embodiments, the kit further comprises instructions for usein the treatment of cancer or infectious disease (e.g., using the liquidformulations of the invention alone or in combination with anotherprophylactic or therapeutic agent), as well as side effects and dosageinformation for one or more routes of administration. Optionallyassociated with such container(s) is a notice in the form prescribed bya governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same.

The invention is further defined by reference to the following examples,which are not meant to limit the scope of the present invention. It willbe apparent to those skilled in the art that many modifications, both tothe materials and methods, may be practiced without departing from thepurpose and interest of the invention.

5.5 EXAMPLES

The following are specific examples of stable aqueous and lyophilizedformulations of a benzo[b]azepine TLR agonist of the present invention.The specific benzo[b]azepine TLR agonist used in these examples is (1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamide.As described herein, a benzo[b]azepine TLR agonist may be formulated atconcentrations of up to 40-50 mg/ml using β-cyclodextrin, preferablysulfobutylether β-cyclodextrin. Specifically, the examples describeformulations of the benzo[b]azepine TLR agonist using either 15% or 25%w/v sulfobutylether β-cyclodextrin. These formulations are stable atroom temperature and at normal refrigeration temperatures. The aqueousformulation is also suitable for administration by subcutaneousinjection. The aqueous formulation may also be lyophilized. An exemplarylyophilization procedure is described below. The 15% sulfobutyletherβ-cyclodextrin formulation is preferred for administration bysubcutaneous injection following reconstitution of the lyophilizedproduct.

5.5.1 Aqueous Formulation

A 40 mg/mL solution of a benzo[b]azepine TLR agonist is prepared asdescribed below. Briefly, the required amount of the agonist isdissolved in 10 mM citrate, pH 2.8-3.2, containing either 25% or 15% w/vsulfobutylether β-cyclodextrin and stirred for 30 minutes. Followingsolubilization, the pH of the solution is adjusted to 6.5, the solutionis stirred for an additional 15 minutes and filtered through a 0.2micron polyethersulfone filter. This formulation is suitable forsubcutaneous injection.

TABLE 1 Stability of aqueous formulation Concentration of TLR Agonist(mg/ml) Target: 40 mg/ml Target: 50 mg/ml Time (days) RT 2-8° C. RT 2-8°C. 0 38.2 43.8 2 40.5 41.4 48.4 49.2 6 37.7 40.8 46.9 47.5 7 38.2 40.846.2 45.6 9 37.5 40.6 46.3 47.4 Formulations were prepared by in-processpH manipulation and filtered as described below. The filtered solutionswere stored at room temperature (RT, about 23° C.) and at 2-8° C. Storedsamples were removed from storage at the indicated time points andevaluated for concentration of the benzo[b]azepine TLR agonist by HPLC.

Detailed Procedure for Aqueous Formulation of benzo[b]azepine TLRAgonist

Weigh out an amount of sulfobutylether β-cyclodextrin required for a 25%w/v solution in the final volume of the formulation.

Weigh out an amount of citric acid required for a 0.19% w/v solution inthe final volume of the formulation.

Weigh out an amount of VTX-378 required for a 40 mg/mL solution in thefinal volume of the formulation.

Transfer a volume of sterile filtered water (0.2 μm) equal to 75% of thebatch volume into the container for batch manufacture. Add the citricacid and sulfobutylether β-cyclodextrin to the container for batchmanufacture and stir the components at medium speed until the citricacid and sulfobutylether β-cyclodextrin dissolve completely.

Add benzo[b]azepine TLR agonist to the above solution and stir for anadditional 15 min.

Adjust the pH of the formulation to 2.8-3.0 using 6N hydrochloric acidand stir at medium speed for 30 min to ensure dissolution ofbenzo[b]azepine TLR agonist in the medium.

Adjust the pH of the formulation to 6.3-6.7 using 6N sodium hydroxideand stir at medium speed for 15 min. Turbidity will be observed in theformulation in this pH range.

Adjust the volume of the formulation to the final volume of the batchusing water and stir at medium speed for 10 min. Ensure that the pH isat 6.3-6.7.

Filter the entire batch into a container (previously rinsed with 0.1 Nsodium hydroxide, 0.2 μm filtered water and filtered 70% ethanol, inthat order, and dried through a sterile 0.2 μm polyethersulfone filteron a clean bench.

If desired, transfer aliquots of the filtered formulation into suitableglass containers (previously rinsed with 0.1 N sodium hydroxide, 0.2 μmfiltered water and filtered 70% ethanol, in that order, and dried) usingsterile disposable pipets on a clean bench.

It is recommended that the formulation be stored at 2-8° C.

5.5.2 Lyophilized Formulation

The 40 mg/mL formulation described above is lyophilized according to thefollowing freeze-drying cycle:

Shelf Load Temperature=−50° C.

Freezing: Hold at −50° C. for 120 min; vacuum to seal chamber

Condenser and Vacuum: Condenser temperature (the temperature of thecondenser at which vacuum will be enabled)=−50° C.; Heat start pressure(the pressure at which the process will continue to primary drying)=400mTorr

Primary Drying: Ramp to 0° C. in 120 min at a vacuum of 100 mTorr; Rampto 10° C. in 80 min at a vacuum of 100 mTorr; Hold at 10° C. for 1920min at a vacuum of 100 mTorr

The lyophilized formulations prepared as described above were stable atboth room temperature (about 23° C.) and at 2-8° C. for at least twomonths as shown in the table below. Samples were removed from storage atthe indicated time point, reconstituted in 0.8 ml of water each, andevaluated for pH, osmolality, and concentration of the benzo[b]azepineTLR agonist by HPLC.

TABLE 2 Stability of lyophilized formulation Time Storage TempOsmolality Conc. of TLR agonist (weeks) (° C.) pH (mOsm/kg) (mg/ml)initial NA 6.39 733 39.9 2 RT 6.35 725 44.0 2-8 6.37 736 39.5 4 RT 6.35722 41.7 2-8 6.34 722 40.1 8 RT 6.37 726 44.0 2-8 6.37 730 44.0Solutions of 40 mg/ml benzo[b]azepine TLR agonist prepared in 15% w/vsulfobutylether β-cyclodextrin (in 10 mM citrate, pH 6.5) werelyophilized as described herein. The lyophilized samples were stored atroom temperature (about 23° C.) and at 2-8° C. for up to 2 months.Samples were removed from storage at the indicated time point,reconstituted in 0.8 ml of water each, and evaluated for pH, osmolality,and concentration of the benzo[b]azepine TLR agonist by HPLC.

Detailed Procedure for Lyophilized Formulation of benzo[b]azepine TLRAgonist

Weigh out the target amount of sulfobutylether β-cyclodextrin requiredfor a 15% w/v solution in the final volume of the formulation.

Weigh out the target amount of citric acid required for a 0.19% w/vsolution in the final volume of the formulation.

Weigh out the target amount of benzo[b]azepine TLR agonist required fora 40 mg/mL solution in the final volume of the formulation.

Transfer a volume of sterile filtered water (0.2 μm) equal to 75% of thebatch volume into the container for batch manufacture. Add the citricacid and sulfobutylether β-cyclodextrin to the container for batchmanufacture and stir the components at medium speed until the citricacid and sulfobutylether β-cyclodextrin dissolve completely.

Add benzo[b]azepine TLR agonist to the above solution and stir for anadditional 30 min.

Adjust the pH of the formulation to 2.8-3.0 by adding 6N hydrochloricacid drop-wise at a rate of approximately 0.5 mL/min allowing thesolution to stir for 1 min at the end of each addition of 0.5 mL. Duringthe drop-wise addition monitor the pH continuously. Once at the desiredpH, continue stirring the solution at medium speed for 30 min to ensuredissolution of benzo[b]azepine TLR agonist in the medium.

Adjust the pH of the formulation to 6.3-6.7 by adding 6N sodiumhydroxide drop-wise at rate of approximately 0.5 mL/min allowing thesolution to stir for 2 min at the end of each addition of 0.5 mL. Duringthe drop-wise addition monitor the pH continuously. Ensure that thetarget pH is reached and do not let the solution pH to reach valuesgreater than 6.7. Once at the desired pH, continue stirring at mediumspeed for 15 min. Turbidity will be observed in the formulation in thispH range.

The volume of each of 6N hydrochloric acid and 6N sodium hydroxide perincremental addition (0.5 mL) mentioned above is based on a batch sizeof 1 L. These volumes should be adjusted proportionally to the finalbatch size, e.g., for a 10 L batch the volume of each addition ofacidlbase will be 5 mL.

Adjust the volume of the formulation to the final volume of the batchusing water and stir at medium speed for 10 min. Check that the pH is at6.3-6.7 and adjust the pH to be within this range, if necessary, using6N sodium hydroxide or 6N hydrochloric acid.

Filter the entire batch into a container (previously rinsed with 0.1 Nsodium hydroxide, 0.2 μm filtered water and filtered 70% ethanol, inthat order, and dried) through a sterile 0.2 μm polyethersulfone filteron a clean bench.

Transfer 1.2 mL aliquots of the filtered formulation into 3-mL serumvials (previously rinsed with 0.1 N sodium hydroxide, 0.2 μm filteredwater and filtered 70% ethanol, in that order, and dried) using steriledisposable pipets on a clean bench.

Transfer the filled vials to the lyophilizer shelf with the grey butylstoppers placed loosely on top of the vials to allow gas flow in and outof the vials through the spaces between the prongs of the stoppers. Usethe lyophilization cycle shown in Attachment 1. Use high purity nitrogento adjust the chamber pressure.

Upon completion of the lyophilization, release the vacuum to dry highpurity nitrogen. Stopper the vials and seal the vials with aluminumcrimp seals.

It is recommended that the formulation be stored at 2-8° C.

5.5.3 Stability of Inclusion Complex Formulation Versus Co-solventFormulation

benzo[b]azepine TLR agonist solutions were prepared either as aninclusion complex formulation or as a co-solvent formulation andevaluated for short-term stability as measured by concentration ofbenzo[b]azepine TLR agonist remaining after storage at the indicatedtemperature for the indicated time.

Inclusion Complex Formulation

A 5 mg/mL benzo[b]azepine TLR agonist solution was prepared in 25% w/vCAPTISOL in 10 mM citrate (pH 6.5). Briefly, the required amounts ofCAPTISOL and citric acid were dissolved in a volume equivalent to ˜75%of batch size. The target amount of benzo[b]azepine TLR agonist wasadded to this solution and stirred for 30 min. The pH of the sample wasadjusted to ˜3.0 using 6N HCl and stirred for 30 min. The pH wasadjusted to ˜6.5 using 6N NaOH and stirred for an additional 15 min. Thevolume of the formulation was made up to 100% of batch size using water.The formulation was then filtered through 0.2 μm polyethersulfone filterand aseptically filled into a sterile container (235 μL per container).All steps of the formulation preparation were carried out at roomtemperature.

Co-solvent Formulation

A 5 mg/mL benzo[b]azepine TLR agonist solution was prepared in a solventsystem containing 10% propylene glycol, 10% polyethylene glycol 400 (PEG400) and 10 mM citrate in water (pH 6.5). Briefly, the required amountof citric acid was dissolved in a volume equivalent to ˜50% of batchsize. The required amounts of propylene glycol and PEG 400 were addedand stirred to mix. The target amount of benzo[b]azepine TLR agonist wasadded to this solution and stirred for 30 min. The pH of the sample wasadjusted to ˜3.0 using 6N HCl and stirred for 30 min. The pH wasadjusted to ˜6.5 using 6N NaOH and stirred for an additional 15 min. Thevolume of the formulation was made up to 100% of batch size using water.The formulation was then filtered through 0.2 μm polyethersulfone filterand aseptically filled into sterile containers (235 μL per container).All steps of the formulation preparation were carried out at roomtemperature.

Stability Evaluation

The containers filled with the inclusion complex formulation were storedeither at room temperature (RT) or at 2-8° C. The containers filled withthe co-solvent formulation were stored either at room temperature, at2-8° C., or at 40° C. The samples were removed from storage at specifictime-points and evaluated for benzo[b]azepine TLR agonist concentrationby HPLC according to standard methods.

Results

Table 3 provides the assay concentration of the benzo[b]azepine TLRagonist in the stability samples at specific time-points and as afunction of storage temperature. As shown in the table, the inclusioncomplex formulation did not show any significant loss of benzo[b]azepineTLR agonist up to 19 days of storage even at room temperature. Incontrast, a significant loss of the benzo[b]azepine TLR agonist wasobserved in the co-solvent formulation after 14 days of storage at roomtemperature (˜29% loss). The degradation of the benzo[b]azepine TLRagonist in the co-solvent formulation was more pronounced at 40° C. Itshould be noted that the co-solvent formulation samples stored at 40° C.and at room temperature contained insoluble material after 7 days and 14days, respectively. Based on these stability studies, inclusion complexformulation prepared with CAPTISOL results in better stability comparedto the co-solvent formulation.

TABLE 3 Stability of benzo[b]azepine TLR agonist formulations formulatedeither as an inclusion complex in CAPTISOL or with a co-solvent (10%propylene glycol, 10% polyethylene glycol 400) VTX-378 concentration(mg/mL) Inclusion Complex Co-solvent Formulation Formulation Time-point2-8° C. RT Time-point 2-8° C. RT 40° C. Initial 5.1 5.1 Initial 4.9 4.94.9  7 days 5.0 5.0  7 days 4.9 4.6 3.2 17 days 5.2 5.0 14 days 4.7 3.52.0 Stability of benzo[b]azepine TLR agonist formulations. Inclusioncomplex and co-solvent formulations of the agonist were evaluated forstability by determining agonist concentration at specific time-pointsfollowing storage at 2-8° C. and at RT (and 40° C. for co-solventformulation).

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims. It will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the invention encompassed bythe appended claims.

We claim:
 1. A stable liquid formulation comprising a Toll-like receptor(TLR) agonist and a β-cyclodextrin, wherein said agonist is(1E,4E)-2-amino-N,N-dipropyl-8-(4-(pyrrolidine-1-carbonyl)phenyl)-3H-benzo[b]azepine-4-carboxamideor a pharmaceutically acceptable salt thereof, and the formulation is anaqueous solution comprising the agonist at a concentration of from about1 mg/ml to about 40 mg/ml and the β-cyclodextrin at a concentration offrom about 1% to about 30% weight/volume, and the pH of the formulationis from about 5.0 to about
 7. 2. The formulation of claim 1, wherein theβ-cyclodextrin is sulfobutyl ether β-cyclodextrin.
 3. The formulation ofclaim 1, wherein the formulation comprises from about 5%-15%weight/volume of the β-cyclodextrin.
 4. The formulation of claim 1,wherein the agonist is formulated at a concentration of from about 2mg/ml to about 15 mg/ml.
 5. A method for treating cancer in a subjectcomprising administering to the subject a stable formulation of claim 1,and the cancer is selected from colon carcinoma, ovarian cancer, breastcancer, head and neck cancer, renal cancer, bladder cancer,hepatocellular cancer and lymphoma.
 6. The method of claim 5, whereinthe TLR agonist is formulated at a concentration of at least 2 mg/ml. 7.The method of claim 5, wherein the formulation is suitable foradministration to the subject by injection.
 8. The method of claim 5,wherein the TLR agonist is administered to the subject at weekly orbiweekly intervals.
 9. A method for treating an infectious disease in asubject comprising administering to the subject a stable formulation ofclaim
 1. 10. A method for treating atopic dermatitis in a subjectcomprising administering to the subject a stable formulation of claim 1.11. A pharmaceutical pack or kit comprising one or more containersfilled with a stable formulation of claim
 1. 12. The formulation ofclaim 1, wherein the formulation is stable at about 20-30° C. atconcentrations up to 40 mg/ml for at least 2 weeks.
 13. The formulationof claim 1, wherein the pH is about 6.5.
 14. The formulation of claim 1,wherein the formulation is an aqueous solution comprising the agonist ata concentration of from about 2 mg/ml to about 15 mg/ml and theβ-cyclodextrin at a concentration of from about 5% to about 15%weight/volume, and the pH of the formulation is from about 5.0 to about7.
 15. A method for treating cancer in a subject in need thereofcomprising administering to the subject the formulation of claim 14,wherein the cancer is selected from colon carcinoma, ovarian cancer,breast cancer, head and neck cancer, renal cancer, bladder cancer,hepatocellular cancer and lymphoma.